Expression, characterization, and structure analysis of recombinant cold-adapted aminopeptidase P from Antarctic Pseudomonas sp. AMS3

Microbial enzymes are increasingly favored in industry due to their superior properties. These include high stability, efficient catalytic activity, and ease of production and optimization compared to enzymes from plants and animals. Proteases, also known as peptidases, are enzymes that break down proteins into smaller amino acids and peptides. One specific type is aminopeptidase P (APPro), a metalloprotease that cleaves the N-terminal amino acid residue from protein chains only when the second residue is proline. Due to its unique ability to cleave proline-containing peptides, APPro finds applications beyond its role in biological pathways. It contributes to various fields, including food science for example eliminating bitterness in cheese and environmental remediation by degrading organophosphorus compounds found in pesticides and chemical warfare agents. While microbial enzymes offer numerous advantages, most research focuses on enzymes functioning at optimal temperatures. However, cold-adapted APPro enzymes, with their ability to function efficiently at low temperatures, hold immense potential for cost￾effective industrial processes. Nevertheless, research on these unique biocatalysts remains scarce. Therefore, in order to better understand the biocatalytic potential of these unique cold-adapted enzymes, in-depth studies of their production, biochemical characterization and structure analysis are crucial. The current study is to express, characterize and analyze the structure through computational and experimental approaches. APPro gene (pepP), from an Antarctic Pseudomonas sp. AMS3 consisting of 1225 nucleotides encoding 407 amino acids was successfully cloned into an expression vector pET32b(+) and expressed in Escherichia coli strain Rosetta-Gami B (DE3). The recombinant enzyme, AMS3-APPro was expressed at optimal conditions of 25°C, 0.25 mM IPTG (inducer) and 16 h post-induction time with a predicted molecular mass of 63 kDa including purification tag (18 kDa). However, the enzyme was expressed in the form of inclusion bodies and therefore, solubilization and refolding of the enzyme were carried out using 8 M urea. Then, the enzyme was purified via two step purifications through Ni-Sepharose affinity chromatography and anion exchange chromatography. Biochemical characterization of AMS3-APPro showed an optimal and stable temperature profile around 20-30°C highlighting its potential as a cold-adapted enzyme. The enzyme also demonstrated high catalytic activity at pH 8. Elevated enzyme activity was also observed in the presence of Mn2+ ion. The structure of the enzyme was proposed best using YASARA software showing a tetrameric structure. The enzyme displayed a two-domain organization, with an N-domain and a C-domain adopting the characteristic pita-bread fold essential for its catalytic activity. Molecular dynamics (MD) simulations also offer valuable insights into the cold-adapted nature of AMS3-APPro. The simulations reveal the enzyme's ability to maintain its native conformation and stability at low temperatures. This sheds light on the structural and dynamic features that underpin its effective function in cold environments. These findings have significant implications for future biotechnological applications of cold-adapted enzymes.

Text 125116 - Full Text.pdf Download (14MB) Official URL or Download Paper: http://ethesis.upm.edu.my/id/eprint/18812

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